Hypothermia and hyperthermia are cell stressed states resulting from environmental temperature changes, which can abnormally decrease intracellular glutathione (GSH) concentrations and induce apoptosis. As the most abundant intracellular non-protein biothiol, GSH can protect cells from apoptosis. Considering the important roles of GSH in the anti-apoptotic process in cells and in vivo, we strive to develop a powerful chemical tool for the direct detection of GSH concentration changes under temperature stress. Herein, we report a ratiometric fluorescent probe (CyO-Dise) based on a selenium-sulfur exchange reaction for the qualitative and quantitative detection of GSH concentration fluctuations in cells and in vivo. The probe has been successfully used to assess the changes of GSH levels in HepG2 and HL-7702 cells using the stimulations of hypothermia and hyperthermia. In terms of the anti-apoptotic effect of GSH under hypothermic and hyperthermic conditions, human normal liver HL-7702 cells have stronger abilities to fight against temperature stress than human liver carcinoma HepG2 cells. Hypothermia and hyperthermia can also improve the drug resistance of cis-dichlorodiamineplatinum(ii) (DDP)-resistant HepG2/DDP cells. The CyO-Dise probe has been employed to image GSH concentration changes in HepG2 and HepG2/DDP xenografts on nude mice. With the adjuvant therapy effects of hypothermia and hyperthermia, the chemotherapy drug DDP exhibits good ability for the treatment of HepG2 and HepG2/DDP xenografts. The above applications make our probe a potential new candidate for the accurate diagnosis of cancer and efficacy evaluation of treatment.